OBJECTIVE To evaluate the genotoxicity of the chloride intermediate in the synthesis of benproperine phosphate with (quantitative) structure-activity relationship computer method (Q)SAR, and to classify it according to the international Counsil for Harmonisation of Technical Requirements for pharmaceuticals for Human Use(ICH) M7(R1) guidelines, and to establish a method for the determination of the chloride intermediate residue in benproperine phosphate API.

METHODS Hazard Assessment and genotoxic impurity classification of the chloride intermediate were carried out by toxicity prediction software-Toxtree(version 3.1.0.1851, Istituto Superiore di Sanita, Italy), Derek Nexus(Derek Nexus: 6.2.1) and Sarah Nexus(Sarah Nexus: 3.2.1)), and the residue control limit of the chloride intermediate was 9.5μg·g⁻¹ ,which was calculated according to threshold of toxicological concern(TTC). A gas chromatography-mass spectrometry method was developed for the determination of the chloride intermediate residue in benproperine phosphate API. An elastic quartz capillary column with 50% phenyl-methylpolysiloxane as stationary liquid DB-17 MS(30 m×0.25 mm, 0.25 μm) was used as the chromatographic column, the column temperature was programmed temperature ( initially heated at 50 ℃ for 1 min, rose to 220 ℃ at a speed of 30 ℃·min⁻¹, remaining 1 min, finally rose to 280 ℃ at a speed of 10 ℃·min⁻¹ remaining 1 min) .The injector temperature was 280 ℃, splitless. The carrier gas was helium, the flow rate was 1.0 ml·min⁻¹, the sample volume was 1 μL, The ion source was electron impact ionization source, the ion source temperature was 250 ℃, the ionization energy was 70 eV, Transmission line temperature was 250 ℃, the scan mode was selective ion monitoring(SIM), the selective ions were m/z 260.5, m/z 165.3, m/z 183.3, m/z 106.2, m/z 260.5 was the quantitative ion quantified by the external reference method.

RESULTS Based on the warning structure of chloro-alkane, Toxtree, Derek Nexus and Sarah Nexus prediction results were positive. Derek and Sarah Software results were based on the fact that alkyl halides were electrophilic compounds which were capable of directly alkylating DNA and could produce free radicals under the action of light or other excitation energies, and that leaded to mutations in DNA. Multiple alkyl halides were mutagenic in the presence and absence of the S9 mixture, particularly against the typhimurium strains TA100 and TA1535. For example, Halomethane and haloethane were a known genotoxic impurity. Halogenated heterocyclic compound, such as 2-(chloromethyl) pyridine, has also been reported to be mutagenic in Ames Test. In conclusion, Derek's prediction that the chloride intermediate was genotoxic was reasonable. Sarah's prediction was based on a well-matched structure of the compound in the database (previously documented) and two supporting structures which showed positive genotoxicity for the chloride intermediate.The Chloride intermediate was classified as class 3 and should be controlled as class 2 potential genotoxic impurity. The established GC-MS method showed a good linear relationship in the concentration range of 1.054−10.54 ng·mL⁻¹, the correlation coefficient was 0.9980, the average recovery(n=9) was 95% , and the precision RSD% was 2.41%(n=6) , the detection limit was 0.2108 ng·mL⁻¹, and the quantitative limit was 0.6325 ng·mL⁻¹. The residues of the chloride intermediate were detected in all 10 batches of benproperine phosphate raw materials, and the residue contents were all lower than the TTC control limit.

CONCLUSION The chloride intermediate in the synthesis of benproperine phosphate is classified as class 3 potential genotoxic impurity and should be controlled as class 2 potential genotoxic impurity as assessed by (Q) SAR, the established method is stable and reliable, and it can be used to monitor the residual level of the chloride intermediate in benproperine phosphate API.